PROJECT SUMMARY Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people in the general population and is a leading cause of heart failure and sudden cardiac death. Despite the significant clinical impact and ongoing basic and translational research, the molecular mechanisms leading to disease onset and progression are poorly understood. Until now, no specific therapeutic approach has been established. In vitro modeling of cardiovascular diseases is of high interest as human adult cardiomyocytes are difficult to isolate and propagate long-term in culture, and animal models have often proven non-predictive of human pathophysiology. Patient- specific human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and genome editing represent novel technologies for modeling cardiomyopathies. In the past funding period, we were able to recruit ~150 HCM patients. Preliminary data shows diastolic dysfunction and aberrant calcium handling in iPSC-CMs with mutations in myosin binding protein C3 (MYBPC3). Furthermore, preliminary data shows a molecular phenotype in MYBPC3 iPSC-CMs characterized by an activation of non-sense mediated decay (NMD) pathway. However, a major limitation of iPSC-CMs is their immature state as well as the non- physiological conditions of in vitro assays used so far. Here we propose to increase the significance of modeling HCM by i) incorporating 2D micropatterned iPSC-CMs and 3D engineered heart tissues (EHTs) to enhance cellular maturation; by ii) co-culturing iPSC-CMs with iPSC-derived endothelial cells (iPSC-ECs) and cardiac fibroblasts (iPSC-CFs) in 3D EHTs, and by (iii) using single cell RNA-seq and proteomics approaches to elucidate molecular mechanisms of HCM cellular crosstalk. Finally, we will evaluate the significance of the NMD pathway in HCM pathogenesis by using the CRISPR-interference (CRISPRi) and CRISPR-activation (CRISPa) technology, which allows us to screen expeditiously the functional relevance of inhibition or activation of a set of genes followed by downstream functional evaluation to localize the target genes with therapeutic potentials.